Expression of SDF-1/CXCR4 and related inflammatory factors in sodium fluoride-treated hepatocytes

At present, the mechanism of fluorosis-induced damage to the hepatic system is unclear. Studies have shown that excess fluoride causes some degree of damage to the liver, including inflammation. The SDF-1/CXCR4 signaling axis has been reported to have an impact on the regulation of inflammation in human cells. In this study, we investigated the role of the SDF-1/CXCR4 signaling axis and related inflammatory factors in fluorosis through in vitro experiments on human hepatic astrocytes (LX-2) cultured with sodium fluoride. CCK-8 assays showed that the median lethal dose at 24 h was 2 mmol/l NaF, and these conditions were used for subsequent enzyme-linked immunosorbent assays (ELISAs) and quantitative real-time polymerase chain reaction (qPCR) analysis. The protein expression levels of SDF-1/CXCR4 and the related inflammatory factors nuclear factor-κB (NF-κB), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) were detected by ELISAs from the experimental and control groups. The mRNA expression levels of these inflammatory indicators were also determined by qPCR in both groups. Moreover, the expression levels of these factors were significantly higher in the experimental group than in the control group at both the protein and mRNA levels (P < 0.05). Excess fluorine may stimulate the SDF-1/CXCR4 signaling axis, activating the inflammatory NF-κB signaling pathway and increasing the expression levels of the related inflammatory factors IL-6, TNF-α and IL-1β. Identification of this mechanism is important for elucidating the pathogenesis of fluorosis-induced liver injury.

. In contrast, drinking water fluorosis is still one of the most severely damaging types of fluorosis in China [5].In highly fluoridated areas, local populations have experienced some degree of bone damage due to drinking water with a high fluoride content [6].Coalburning fluorosis is also common in China, mainly in the southwest [7,8].Approximately 43% of the districts and counties in Guizhou Province of China were reported to be impacted by fluorosis, and more than half of the individuals in these areas suffer from fluorosis [9].With the global economic development and changes in industry and lifestyle, the prevalence of endemic fluorosis has been significantly reduced, but groundwater fluoride contamination and tea-drinking fluorosis are still prevalent in some areas [10][11][12][13].Thus, fluorosis remains a global public health problem that cannot be ignored, and effective control of fluorosis is important for improving quality of life.
Fluorine is an essential trace element that often exists in natural compounds, and it can be bioactive in humans.The intake of an appropriate amount of fluorine is beneficial, for example, for caries prevention and treatment [14].Excessive intake of elemental fluoride, however, can cause damage to bones and other important organs in the body [15], such as the cardiovascular and hepatic systems and teeth [16][17][18][19].According to epidemiological investigations and in vitro and in vivo experimental studies, fluorosis may cause a certain degree of damage to the liver, inducing hepatic glucose and lipid metabolic dysfunction, and at the cellular level, fluorosis can cause swelling of the endoplasmic reticulum and mitochondria, reduced nuclear volume, nuclear membrane wrinkling and other defects in hepatocytes [20,21].Fluorosis was shown to increase the levels of inflammatory factors [22].However, the pathogenesis of the resulting liver inflammation is unclear.
Currently, signaling pathways are a hot topic in the study of the pathogenesis of fluorosis.
The stromal cell-derived factor 1 (SDF-1) and chemokine receptor 4 (CXCR4) signaling axis is important for regulating inflammation in the human body [23][24][25].CXCR4 is a member of the G protein-coupled receptor superfamily and, along with its specific ligand SDF-1, also known as C-X-C motif chemokine ligand 12, forms a chemokine network that is involved in physiological processes such as cellular immunity, inflammatory cell metastasis, and cell proliferation in humans.Chemokines are a large group of small molecule inflammatory cytokines with molecular weights in the range of 8-12 kDa that act by binding to the corresponding G-protein-coupled seven-transmembrane receptors present on the surface of the target cells to perform various physiological functions [26][27][28].The expression of SDF-1/CXCR4 signaling axis members has been reported to be significantly higher in the serum of patients with osteofluorosis than in that of controls, and the expression level of the inflammatory factor NF-κB was positively correlated with chemokine levels [29].Activation of the NF-κB pathway is closely linked to inflammatory mechanisms in disease [30][31][32].The In this study, LX-2 cells were cultured with sodium fluoride for in vitro experiments, and cell survival was evaluated by the CCK-8 method.HE staining was used to observe the growth and morphology of the cells in each group, and the expression levels of SDF-1 and CXCR4, as well as the inflammatory factors related to the SDF-1/CXCR4 signaling pathway, such as IL-6, TNF-α, NF-κB, and IL-1β, were analyzed to provide evidence for the mechanism of fluoride intoxication-induced liver inflammation.The results provide a basis for the mechanism of hepatic inflammation caused by fluorosis.

Cell Culture
Human liver astrocytes (LX-2, 1 vial, model T25) were purchased from Wuhan Procell Life Sciences Co.The cells were cultured in a humidified incubator at 37°C with 5% CO2 for 48 h.
When the cell confluence was greater than 80%, the cells were cultured in a humidified incubator at 37°C with 5% CO2 and rinsed twice with 2 ml of 1x PBS (Thermo Fisher Scientific, China).Then, 1 ml of trypsin-EDTA (Thermo Fisher Scientific, China) was added to digest the adherent cells, and the cells were placed in a 37°C and 5% CO2 incubator for 3 min.Then, 4 ml of LX-2 medium was added to the T25 cell culture flasks (Wuhan Procell Life Sciences Co., Ltd., China) to terminate tryptic digestion and mixed with a sterile pasteurized pipette to resuspend the cells.Then, the cells were transferred to 15 ml sterile and enzyme-free centrifuge tubes (Thermo Fisher Scientific, Ltd., China), centrifuged at 1200 × g for 3 min, and resuspended in an appropriate volume of medium for transfer to new culture flasks incubated at 37°C and 5% CO2 to continue the cultivation of the cell lines.The present study used cells from the 3rd-7th passages.

Sodium Fluoride for Cell Treatments
Sodium fluoride (10.5 g, Tianjin Yong da Chemical Reagent Co., Ltd., China) was dissolved in 500 ml of 1x PBS solution and filtered through a membrane to remove any bacteria, and the concentration was adjusted to 500 mmol/l.Complete media with different NaF concentrations were prepared according to each group, as shown in Table 1.After LX-2 cells were cultured at 37°C in a 5% CO2 incubator for 24 h, the cells were treated with fluorine (n=4).

CCK-8 Assay for Cell Viability
Cells (Nanjing Novozymes Bioscience and Technology Co., Ltd., China) were aliquoted into 96-well plates containing 2,000 cells/ml and incubated for 24 h, and the culture medium was poured off when the cell wall density was greater than 80%.The cells were rinsed twice with PBS, and a blank group, control group (0 mmol/l NaF), and experimental groups with different concentrations of sodium fluoride (0.5, 1, 2, 4, 8 mmol/l NaF) were prepared.For each well, except those for the blank group, 200 µl of cell culture medium was added, and the cells were incubated for 12 h, 24 h or 48 h.Then, 10 µl of CCK-8 reagent was added, and the cells were incubated for 1 h with protection from light.A fully automated enzyme labeling instrument (Thermo Fisher Scientific, China) was used to detect the OD value of each group at 450 nm, and cell viability (%) was calculated as (OD of the experimental wells-OD of the blank wells/OD of the control wells) × 100% (n=4).

HE Staining
LX-2 cell cultures were fixed with 4% paraformaldehyde for 5-10 min, washed with water for 1 min, treated with HD Constant Staining Pretreatment Solution for 1 min, and sequentially stained with appropriate amounts of hematoxylin and eosin staining solution.Finally, the cells were fixed with neutral resin, and the morphology of LX-2 hepatocytes was observed under a light microscope.

Enzyme-Linked Immunosorbent Assays (ELISAs) of the Expression of Each Target Protein in the Cell Supernatant and Cell Extract of Each Group of Cells
CXCR4, SDF-1, IL-1β, TNF-α, IL-6 and NF-κB ELISA kits were obtained from Nanjing Boyan Biotechnology Co., Ltd.(China).Cell supernatant collection: Cell supernatants were collected by centrifugation of each test group at 20 min (3000 r/min) and stored at -20°C to avoid repeated freezing and thawing.Cell extract collection: adherent cells were washed with PBS at 4°C and digested with trypsin, and then, the cells were collected after 5 min (1000 r/min) and washed with PBS 3 times.Then, 200 µl of 1x PBS was added to resuspend the cells, the cells were ruptured by repeated freezing and thawing, and the supernatant was collected for evaluation.The expression level of each target protein in the cell supernatant was detected by an enzyme labeling instrument (450 nm).The kits were left at room temperature for 30 min before use.For the wash buffer, the concentrate in the kit was diluted with distilled water at 1:20.
All reagents and samples were used at room temperature.Blank wells (no sample or standard added), standard wells (50 µl of standards at different concentrations) and sample wells (50 µl of samples to be tested) were prepared, and 2 replicate wells were prepared for each group.Horseradish peroxidase (HRP)-labeled antibody (100 µl) was added to each of the wells.The plate was sealed using plate sealing film and incubated for 60 min at 37°C in a water bath or thermostat.The liquid was discarded, 300 µl of wash solution was added to each well, and the plate was washed for 20 s and patted dry on absorbent paper; the washes were repeated 5 times.Then, 50 µl each of chromatography substrates A and B was added to each well and incubated at 37°C for 15 min.Finally, 50 µl of termination solution was added to each well, the color changed from blue to yellow, and readings were taken within 15 min.

RNA Extraction
Total RNA was extracted with a Total RNA Extraction Kit (All Specialty Gold Biotechnology Co., Ltd., Beijing, China).Then, cDNA was synthesized with a Reverse Transcription Kit (All Specialty Gold Biotechnology Co., Ltd., Beijing, China) for gene fragment detection; gene fragments were amplified, and the optimal primers as determined by the HIFI RCR enzyme (All Specialty Gold Biotechnology Co., Ltd., Beijing, China) and HIFI RCR enzyme (Beijing Total Gold Biotechnology Co., Ltd., Beijing, China) were used to amplify the gene fragments at the optimal primer annealing temperature.A fully automated enzyme labeling instrument (Thermo Fisher Scientific) was used to determine the CXCR4, SDF-1, IL-1β, TNF-α, IL-6, and NF-κB mRNA levels as well as the A260/280 ratio.Agarose electrophoresis buffer (LABJIC Biotechnology Co., Ltd., Beijing, China) was used to prepare a 1% agarose gel to determine the RNA concentration.

Real-Time Quantitative Polymerase Chain Reaction (qPCR)
Each reaction contained 0.4 µl of upstream primer, 0.4 µl of related primer, 0. and exhibited homogeneity of variance, and the Mann-Whitney test was used for data that did not satisfy these conditions.Measurement data are expressed as the mean ± standard deviation (± s), test level α = 0.05, and P < 0.05 indicated significance.

Effects of Different Exposure Times and Sodium Fluoride Concentrations on the Survival of Human LX-2 Cells
We first investigated the effects of different concentrations of sodium fluoride and different fluoride staining times on cell survival by using CCK-8 assays.The results showed that with different concentrations (0, 0.5, 1, 2, 4 and 8 mmol/l) of NaF, the survival rate of the cells gradually decreased with increasing fluoride concentration, and the LD50 values for LX-2 cells at 12 h, 24 h and 48 h were all approximately 2 mmol/l NaF (see Figure 1).Therefore, 2 mmol/l NaF was selected as the treatment concentration for the subsequent experiments.The above figure shows the morphology of normal hepatocytes and sodium fluoridetreated LX-2 hepatocytes under a light microscope.Sodium fluoride was added at a concentration of 2 mmol/l for a period of 24 h.The cells in the fluoride-treated group were relatively rounded or swollen; moreover, the nuclei were more centralized, and the cytoplasm was dramatically smaller in volume.Finally, the cellular arrangement was disorganized, and the interstitial space between the cells was enlarged, among other phenomena (see Figure 2).

Increased Expression of Inflammatory Factors in the Supernatants of NaF-Treated Hepatocytes
The OD values of the experimental group and the control group were determined at 450 nm by an enzyme marker, and t tests were used to identify any difference between the OD values of the two groups.ELISA results showed that the expression of CXCR4, SDF-1, IL-1β, TNF-α, IL-6 and NF-κB-specific proteins was significantly increased in the fluoride-treated cell group compared to the control group (P < 0.05), as shown in Table 3 and Figure 3.

Increased Expression of Inflammatory Factors in NaF-Treated Hepatocyte Extracts
ELISAs of cell extracts showed that CXCR4, SDF-1 (CXCL12), IL-1β, TNF-α, IL-6, and NF-κB protein expression was significantly increased in the fluoride-treated cell group compared to the control group (P < 0.05), as shown in Table 4 and Figure 4.  Cell extracts were mainly obtained by repeated freezing and thawing of the cells, and the lysates were centrifuged to determine whether inflammatory factor receptors were expressed in the cells.Cell supernatants were directly centrifuged and used to determine whether inflammatory factor receptors were expressed on the surface of the cell membranes.ELISAs of both the cell extracts and the cell supernatants showed that fluoride significantly increased the expression of cellular inflammatory factors at this concentration.

Analysis of qPCR Results
Fluorescent dyes were added to the PCR system, and fluorescent signal accumulatio n was used to monitor the PCR process.We used GAPDH as the internal reference gen e, and the 2-ΔΔCT method was used to calculate the mRNA levels of CXCR4, SDF-1, IL -1β, TNF-α, IL-6 and NF-κB, which were determined by qPCR in the fluoride group an d the control group, as shown in Figure 5.
We concluded that fluoride enhanced the SDF-1/CXCR4 signaling axis and the inflammatory factors IL-1β, TNF-α, IL-6 and NF-κB in normal hepatocytes, both in terms of gene expression and protein expression, as shown by ELISAs and qPCR.

Discussion
Fluorosis occurs in many regions of the world and affects human quality of life to a certain extent, not only by placing an economic burden on families but also by impeding the economic development of a region [39].However, the pathogenesis of fluorosis has not yet been clarified, although it is now known that fluorosis mainly involves oxidative stress, endoplasmic reticulum stress, and signaling pathways that damage human tissues or organs [40][41][42].
Although inflammatory damage is not widely recognized as a major component of fluorosis, fluorosis has been shown to cause increased expression of inflammatory factors such as IL-6 [43,44].Thus, inflammatory damage may be one of the pathogenic mechanisms leading to fluorosis, but whether it plays a role in specific cases needs to be evaluated on a case-by-case basis.SDF-1/CXCR4, as an important signaling pathway, recruits cellular immune factors to participate in the body's inflammatory response [45][46].In this study, human LX-2 cells were treated with different concentrations of NaF in vitro, and the cell survival rate was detected by the CCK-8 method.The LD50 value, which was found to be 2 mmol/l after fluoridation with different concentrations of NaF (0.5, 1, 2, 4, and 8 mmol/l) for 12, 24, and 48 h, was selected as the appropriate concentration for further experiments.We analyzed the morphology of the cells before and after fluoride treatment, as well as the mRNA and protein expression of each target, CXCR4, CXCL12, NF-κB, IL-6, IL-1β and TNF-α.HE staining revealed that the cells in the fluoride-treated group exhibited a rounded or swollen morphology, with more compact nuclei; moreover, the arrangement of the cells was disorganized, and the intercellular gap was enlarged.HE staining showed that excessive fluorine caused a certain degree of damage to normal human hepatocytes.ELISAs showed that the levels of CXCR4, CXCL12, NF-κB, IL-6, IL-1β and TNF-α in the cell supernatants and cell extracts were significantly elevated in the experimental group compared with the control group (P<0.05).qPCR was used to determine the mRNA levels of the target genes CXCR4, CXCL12, NF-κB, IL-6, IL-1β and TNF-α.TNF-α mRNA expression levels were significantly increased in the experimental group compared with the control group (P < 0.05), consistent with the ELISA results.
In the present study, one limitation is that we did not explore the relationship between the signaling axis and inflammatory factors, which needs to be explored by signaling pathway antagonist experiments.However, the present cellular experiments successfully identified the SDF-1/CXCR4 signaling axis, and the related inflammatory factors NF-κB, IL-6, IL-1β, and TNFα were expressed at high levels in hepatocytes treated with high concentrations of fluoride.We found that the gene and protein expression levels of SDF-1/CXCR4 signaling axis members in the fluoride group were significantly higher than those in the blank control group, and the mRNA expression levels of inflammatory molecules such as NF-κB, IL-6, IL-1β, and TNF-α were consistent with those of the SDF-1/CXCR4 signaling axis in both groups.We speculated that fluoride could lead to upregulated expression of genes and proteins of the SDF-1/CXCR4 signaling axis in hepatocytes and then activate the NF-κB signaling pathway to release excessive inflammatory factors such as IL-6, IL-1β and TNF-α.The association between the pathogenesis of inflammation in fluorosis and signaling pathways may be confirmed by further investigating signaling pathway antagonists that block signaling pathways to explore the expression of inflammatory factors.As the factors that cause inflammation are very complex, the present findings may also suggest the synergistic activation of the NF-κB signaling pathway and other signaling pathways, which are involved in the release of the inflammatory factors IL-6, IL-1β, and TNF-α [48].The mechanism needs to be further studied.

Conclusion
Excessive sodium fluoride induced an increase in the expression levels of the hepatic cellular inflammatory factors IL-6, TNF-α and IL-1β along with the chemokine signaling axis SDF-1/CXCR4 and the inflammatory signaling pathway NF-κB.

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typical NF-κB pathway induces the production of proinflammatory cytokines such as TNF-α and IL-1β in the innate immune system to mediate inflammatory responses.In addition, the SDF-1/CXCR4 signaling axis was shown to activate the NF-κB signaling pathway to participate in the inflammatory response[33][34][35], suggesting that the SDF-1/CXCR4-NF-κB signaling pathway may play a role in the pathogenesis of fluorosis.Both the SDF-1/CXCR4 signaling axis and the NF-κB signaling pathway can recruit and mobilize inflammatory factors, which may act as signature downstream signaling molecules[36][37][38].The mechanism of the SDF-1/CXCR4 signaling axis and its related inflammatory factors NF-κB, IL-6, IL-1β and TNF-α in the context of the pathogenesis of fluorosis has been poorly studied, and differences in the expression of the above proteins have been reported only in serum samples from individuals in areas with fluorosis[29].Therefore, the present study aimed to further investigate the role of the CXCL12/CXCR4 signaling axis and the related inflammatory factors IL-1β, TNF-α, IL-6 and NF-κB in the mechanism of fluorosis-induced liver injury at the cellular level.Although inflammatory damage has not been conclusively established in the pathogenesis of fluorosis, several studies have shown a correlation between these factors.Whether inflammatory factors such as SDF-1/CXCR4, IL-6, TNF-α, NF-κB, and IL-1β play a role in the hepatic system due to fluorosis deserves further exploration.

Figure 1 .
Figure 1.Effects of sodium fluoride exposure time and concentration on the viability of LX-2

Figure 2 .
Figure 2. Results of HE staining of cells (n=3)

Figure 3 .
Figure 3. Expression level of each target protein in the supernatants of the fluoride-treated

Figure 4 .Table 4 .
Figure 4. Expression level of each target protein in cell extracts from the fluoride-treated and

Figure 5 .
Figure 5. mRNA expression levels of indicators in the fluoride group and the control group

Table 1 .
Configuration Table for Different NaF Concentrations

Table 2 .
TNF-α, IL-6 and NF-κB was calculated by the 2 -ΔΔCT method using glyceraldehyde dehydrogenase 3-phosphate (GAPDH) as the internal reference gene (n=6).Primer sequences, gene numbers and product amplification lengths Excel was used for data entry and the development of statistical tables, GraphPad Prism 8.0.2 was used to generate statistical graphs, and SPSS 22.0 was used to statistically analyze the experimental and control groups; a t test was used for data that were normally distributed 10 s (44 cycles).The experimental results were obtained by repeating the experiment three times and taking the average value.The mRNA expression of the target genes CXCR4, SDF-1, IL-1β, Note: a P < 0.05 compared to the same indicator in the control group, t Contributions: Conceptualization, R.Y.; methodology, R.Y.; software, R.Y.; validation, R.Y. and H.S.; formal analysis, R.Y. and H.S.; investigation, R.Y., G.P. and R.Y.; data curation, Y.L., J.H. and M.W.; writing-original draft preparation, R. Y; writing-review and editing, R.Y.; visualization, R.Y., X.C. and P.C. and J.M. and L.Q.; supervision, R.Y. and Y.Z. and S.Z.; project administration, Q.Z.; funding acquisition, Q.Z.All authors have read and agreed to the published version of the manuscript.
Funding: The Second Comprehensive Scientific Investigation and Research Support of Qinghai Tibet Plateau, grant number 2019QZKK0607.Data Availability Statement: Not applicable.